Alcohol Alizarin Test

The Alcohol Alizarin Test is a rapid quality control method used to assess the stability and acidity of milk, particularly its suitability for heat processing (e.g., pasteurization, UHT, or evaporation). It combines the principles of the Alcohol Precipitation Test with the pH-indicating properties of alizarin dye, providing a dual evaluation of protein stability and milk acidity in a single step. Here’s a detailed breakdown:

Purpose

• Protein Stability: Determines if milk proteins (mainly casein) will coagulate under heat treatment, which is critical for processing.

• Acidity Assessment: Indicates the approximate pH or acidity level, helping identify spoilage or abnormal milk (e.g., from mastitis).

• Quality Screening: Used at farms or dairy platforms to quickly decide if milk should be accepted or rejected.

Principle

• Alcohol Effect: Ethanol dehydrates milk proteins and neutralizes their charge, causing precipitation if the proteins are unstable due to high acidity (e.g., from bacterial fermentation) or disrupted mineral balance (e.g., from mastitis or colostrum).

• Alizarin Effect: Alizarin, a pH-sensitive dye, changes color based on milk’s acidity:

  • Yellow to brownish-yellow: Acidic milk (pH <5.5-6.0).

  • Reddish-brown to lilac: Normal milk (pH 6.6-6.8).

  • Violet to purple: Alkaline milk (pH >7.0, often mastitis-related).

• A positive test (flakes/clots) with color change provides insight into both stability and the underlying cause.

Procedure

1. Sample Collection:

   • Take a small, well-mixed milk sample (e.g., 2-5 mL) from a bulk tank or container.

2. Reagents:

   • Alcohol-alizarin solution: Typically 68-70% ethanol (v/v) with 0.02-0.2% alizarin powder dissolved (exact concentration varies by protocol).

3. Steps:

   • Mix equal volumes of milk and alcohol-alizarin solution (e.g., 2 mL milk + 2 mL solution) in a test tube.

   • Shake gently to combine.

   • Observe immediately (within 1-2 minutes) for precipitation and color change.

4. Observation:

   • Color: Note the hue against a standard chart.

   • Precipitation: Check for flakes, clots, or gel formation.

• Negative Result: No precipitation, lilac/reddish color—milk is stable and suitable for processing.

• Positive Result: Flakes or clots with abnormal color—milk is unstable, likely due to acidity, mastitis, or colostrum.

Factors Causing a Positive Test

1. High Acidity: Lactic acid from bacterial growth (e.g., Lactobacillus) lowers pH, destabilizing proteins.

2. Mastitis: Altered salt balance (higher sodium, lower potassium) and sometimes higher pH reduce stability.

3. Colostrum: High protein content (e.g., immunoglobulins) increases precipitation risk.

4. Mineral Imbalance: Excess calcium/magnesium disrupts casein micelles.

5. Sweet Curdling: Rare bacterial enzymes (e.g., rennin-like) cause clotting without acidity.

Applications

• Farm Level: Screens individual cow or bulk milk for quality issues.

• Platform Testing: At dairy plants, ensures incoming milk won’t curdle during processing.

• Developing Regions: Popular where lab facilities are limited due to its simplicity.

Advantages

• Dual Insight: Combines stability and acidity checks in one test.

• Rapid: Results in 1-2 minutes.

• Simple: Requires only a test tube and reagent, no complex equipment.

Limitations

• Qualitative: Doesn’t quantify exact acidity or microbial load.

• Subjectivity: Color and precipitation interpretation can vary between observers.

• False Positives: Normal milk with slight mineral variations may precipitate at higher alcohol concentrations (e.g., 75-80%).

• Limited Scope: Misses other defects (e.g., antibiotics, off-flavors).

Correlation with Other Tests

• Alcohol Test: Similar precipitation focus, but lacks pH indication.

• COB Test: Confirms heat stability; alcohol-alizarin is more sensitive to early instability.

• pH/Acidity: Color changes align with pH <6.4 or acidity >0.17%.

Practical Example

• Scenario: 2 mL milk + 2 mL 70% alcohol-alizarin solution.

  • Result: Lilac, no flakes—accepted as fresh and stable.

  • Alternative: Yellow, large flakes—rejected as spoiled, high acidity confirmed.